Interaction of IRF9 and STAT2 synergistically up-regulates IFN and PKR transcription in Ctenopharyngodon idella
Interaction of IRF9 and STAT2 synergistically up-regulates IFN and PKR transcription in Ctenopharyngodon idella
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IRF9 和 STAT2 的相互作用协同上调草鱼中 IFN 和 PKR 的转录
DOI：
10.1016/j.molimm.2017.03.013
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发表时间：
2017
影响因子：
3.6
通讯作者：
Hu Chengyu
中科院分区：
文献类型：
--
作者：
Wu Zhen;Wang Liqiang;Xu Xiaowen;Lin Gang;Mao Huiling;Ran Xiaoqin;Zhang Tao;Huang Keyi;Wang Haizhou;Huang Qingli;Xu Qun;Hu Chengyu
IRF 9是JAK-STAT通路中的关键因子。在I型IFN的刺激下，IRF 9与STAT 1和STAT 2相互作用形成IFN-1刺激的基因因子3（ISGF 3），其激活ISG的转录。然而，许多研究还表明，二聚体IRF 9/STAT 2而不是三聚体IRF 9/STAT 1/STAT 2在细胞中响应IFN信号充当ISGF 3。本研究克隆并鉴定了草鱼IRF 9（termCiIRF 9，KT 601055）和STAT 2（termCiSTAT 2，KT 781914）的全长cDNA序列。RT-PCR检测到CiIRF 9在草鱼组织中呈低水平的组成型表达，但在LPS和poly I：C刺激下表达显著上调;体外凝胶迁移率变动分析表明CiIRF 9与CiIFNorCiPKR启动子之间存在高亲和力的相互作用;体内瞬时转染CiIRF 9可显著提高CiIFN和CiPKR启动子的活性，而共转染CiIRF 9和CiSTAT 2可显著提高CiIFN和CiPKR启动子的活性。此外，通过体内和体外蛋白质相互作用测定进一步研究CiIRF 9和CiSTAT 2的相互作用。通过瞬时转染实验，在HEK 293 T细胞中表达了标记有FLAG（或HA）的反义CiIRF 9和CiSTAT 2。免疫共沉淀实验表明，CiIRF 9在体内可与CiSTAT 2相互作用。弗罗姆中表达并纯化了可溶性GST-ST 2 -936（含有CiSTAT 2的N-末端和卷曲螺旋结构域）。杆菌GST下拉测定表明GST标记的ST 2 -936有效结合FLAG标记的IRF 9。结果表明，IRF 9和STAT 2的相互作用协同上调IFN和ISG基因的转录水平。
IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription ofISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence ofIRF9(termedCiIRF9, KT601055) andSTAT2(termCiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression ofCiIRF9was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation.In vitro, a high-affinity interaction betweenCiIRF9 and the promoter ofCiIFNorCiPKRwas demonstrated by gel mobility shift assay.In vivo, the promoter activities ofCiIFNandCiPKRwere not only increased by transient transfection ofCiIRF9, but also prominently increased by co-transfection ofCiIRF9andCiSTAT2. Moreover, the interaction ofCiIRF9 andCiSTAT2 was further investigated byin vivoandin vitroprotein interaction assays. RecombinantCiIRF9 andCiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed thatCiIRF9 can interact withCiSTAT2in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain ofCiSTAT2) was expressed and purified fromE. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level ofIFNandISGgenes.